Proteases and peptidases are enzymes that catalyse the hydrolysis of peptidic amide bonds. Proteases play an important role in the regulation of biological processes in almost every life-form from bacteria to virus to mammals. They perform critical functions in, for example, digestion, blood clotting, apoptosis, activation of immune responses, zymogen activation, viral maturation, protein secretion and protein trafficking. They can be classified according to a number of criteria, such as site of action, substrate preference, and mechanism. So, for example, aminopeptidases act preferentially at the N-terminal residues of a peptide, while carboxypeptidases act preferentially at the C-terminus and endopeptidases act at sites removed from the two termini. Among the carboxy- and aminopeptidases, peptidyl peptidases cleave a single amino acid residue from the substrate, dipeptidyl peptidases cleave a dipeptide unit (two amino acids) from the substrate, and tripeptidases cleave three amino acids from the substrate. Substrate preference is frequently expressed in terms of the amino acid residue immediately N-terminal to the cleavage site. For example, trypsin-like peptidases will preferentially cleave a peptide next to a basic amino acid (arginine or lysine), i.e. where the bond hydrolysed is the Arg/Lys-Xaa bond. As another example, the chymotrypsin-like family of peptidases preferentially hydrolyse peptides adjacent to an aromatic residue. Mechanistically, peptidases are classified as being serine-dependent, cysteine-dependent, aspartic acid-dependent or zinc-dependent.
Because peptidases and proteases are involved in the regulation of many physiological processes, they are attractive targets for the development of therapeutic agents. Protease and peptidase inhibitors are, for example, used in the treatment of hypertension, coagulation disorders, and viral infection.
Proteolytic enzymes that exploit serine in their catalytic activity are ubiquitous, being found in viruses, bacteria and eukaryotes. Over 20 families (denoted S1–S27) of serine protease have been identified; these are grouped into 6 clans (SA, SB, SC, SE, SF and SG) on the basis of structural similarity and other functional evidence. Structures are known for four of the clans (SA, SB, SC and SE); these appear to be totally unrelated, suggesting at least four evolutionary origins of serine peptidases and possibly many more, Rawlings and Barrett, Meth. Enzymol. 244: 19–61 (1994).
The prolyl oligopeptidase family consists of a number of evolutionarily related peptidases whose catalytic activity seems to be provided by a charge relay system similar to that of the trypsin family of serine proteases, but which evolved by independent convergent evolution. A conserved serine residue has been shown experimentally (in E. coli protease II as well as in pig and bacterial PE) to be necessary for the catalytic mechanism. This serine, which is part of the catalytic triad (Ser, His, Asp), is generally located about 150 residues away from the C-terminal extremity of these enzymes (which are all proteins that contains about 700 to 800 amino acids).
One of the most intensively studied prolyl oligopeptidases is dipeptidyl peptidase IV (DPPIV, EC 3.414.5), a type II glycoprotein, which is the only well characterised dipeptidyl aminopeptidase known to be located on the outer side of plasma membranes. As indicated above, dipeptidyl aminopeptidases are characterised by their ability to cleave N-terminal dipeptides from a variety of small peptides. Dipeptidyl aminopeptidases show different substrate specificities and cellular localisation, suggesting different functions of each activity in peptide processing. DPPIV is characterised by its capacity to cleave N-terminal dipeptides containing proline or alanine as the penultimate residue. The DPPIV gene spans approximately 70 kb and contains 26 exons, ranging in size from 45 bp to 1.4 kb. The nucleotide sequence (3,465 bp) of the cDNA contains an open reading frame encoding a polypeptide comprising 766 amino acids. The nucleotides that encode the active site sequence (G-W-S-Y-G) are split between 2 exons. This clearly distinguishes the genomic organisation of the prolyl oligopeptidase family from that of the classic serine protease family.
DPPIV is widely distributed in mammalian tissues and is found in great abundance in the kidney, intestinal epithelium and placenta (Yaron, A. and Naider, F., Critical Reviews in Biochem. Mol. Biol. 1993 [1], 31). In the human immune system, the enzyme is expressed almost exclusively by activated T-lymphocytes of the CD4+ type where the enzyme has been shown to be synonymous with the cell-surface antigen CD26. Although the exact role of DP-IV in human physiology is still not completely understood, recent research has shown that the enzyme clearly has a major role in human physiology and pathophysiology.
On human T cells, DPPIV expression appears late in thymic differentiation and is preferentially restricted to the CD4+ helper/memory population, and CD26 can deliver a potent co-stimulatory T-cell activation signal. DPPIV, also known as T-cell activation antigen CD26, therefore plays an important role in the immune response via association with CD45 tyrosine phosphatase and, through its ability to bind adenosine deaminase (ADA) to the T-cell surface, protects the T-cell from adenosine-mediated inhibition of proliferation. Furthermore, the regulation of the function of chemokines by CD26/DPPIV appears to be essential for lymphocyte trafficking and infectivity of HIV strains. DPPIV has been associated with numerous functions including involvement in T-cell activation, cell adhesion, digestion of proline containing peptides in the kidney and intestines, HIV infection and apoptosis, and regulation of tumorigenicity in certain melanoma cells, Pethiyagoda et al., Clin. Exp. Metastasis 2000;18(5):391–400. DPPIV is also implicated in the endocrine regulation and metabolic physiology. More particularly, DPPIV cleaves the amino-terminal His-Ala dipeptide of GLP-1, generating a GLP-1 receptor antagonist, and thereby shortens the physiological response to GLP-1. Glucagon-like peptide-1 (GLP-1), an incretin that induces glucose-dependent insulin secretion, is rapidly degraded by DPPIV, and since the half-life for DPPIV cleavage is much shorter than the half-life for removal of GLP-1 from circulation, a significant increase in GLP-1 bioactivity (5- to 10-fold) is anticipated from DPP-IV inhibition. Inhibitors of DPPIV are currently being studied in the clinic as potential therapeutic agents for type 2 diabetes and impaired glucose tolerance.
Various different inhibitors of DPPIV were known in 1993. One of these is a suicide inhibitor N-Ala-Pro-O-(nitrobenzoyl-) hydroxylamine. Another is a competitive inhibitor: e-(4-nitro) benzoxycarbonyl-Lys-Pro, and another is a polyclonal rabbit anti-porcine kidney DPPIV immunoglobulin. Others have since been developed and are described in detail in U.S. Pat. Nos. 5,939,560, 6,110,949m 6,011,155 and 5,462,928.
In addition to, but independent of, its serine type catalytic activity, DPPIV binds closely to the soluble extracellular enzyme adenosine deaminase (ADA), acting as a receptor and is thought to mediate signal transduction. DPPIV structure is characterized by two extracellular domains, an α/β fold hydrolase domain and a 7-blade beta-propeller domain consisting of repeated beta sheets of about 50 amino acids. Recently it has been shown that, besides selecting substrates by size, the beta-propeller domain, containing 10 of the 12 highly conserved cysteine residues, contributes to catalysis of the peptidase domain. In addition, the cysteine-rich domain is responsible for DPPIV-binding to collagen I and to extracellular ADA. DPPIV is also reported to play a role in fibronectin-mediated interactions of cells with extracellular matrix. Recent studies show that the protease activity of DPPIV is not required for its anti-invasive activity because mutants of DPPIV that lack the extracellular serine protease activity maintain such activity.
A number of proteins that share similarities with DPPIV have been reported in the literature. Several of these proteins have been cloned including DPP-I, DPP-II, DPP-III, DPP-X and fibroblast activation protein (FAP). These have been identified and characterised either by molecular cloning and functional studies of expressed proteins or as biochemical activities in tissue extracts. DPPIV-beta and other novel peptidases with functional similarities to DPPIV are not yet cloned. The identification, characterization and/or appropriate classification of further members of the family of prolyl oligopeptidases, the elucidation of their physiological (and particularly pathophysiological) role, and the application of that knowledge to the development of new therapeutic agents are significant challenges.